In vitro and in vivo assay of the ER lipid scramblase TMEM41B

Summary Amphipathic phospholipids translocated by scramblases play a central role in facilitating lipid movement across the membrane bilayer, especially at the endoplasmic reticulum (ER) membranes. Here, we present a protocol for assessing the activity of the ER-localized lipid scramblase TMEM41B. We detail an in vitro fluorescent liposome-based phospholipid scrambling assay and in vivo metabolic labeling in living cells using alkyne-choline. The scramblase activity of other VTT (VMP1, TMEM41, and Tvp38) domain-containing proteins, such as TMEM41A and VMP1, can be assayed. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021) .